Method for treating hypocalcemia

ABSTRACT

A method for treating hypoparathyroidism and/or hypocalcemia by local administration of a neurotoxin, such as a botulinum toxin, to a parathyroid gland, thereby reducing an inhibitory effect upon parathyroid hormone secretion. A method for treating hyperparathyroidism and/or hypercalcemia by local administration of a neurotoxin, such as a botulinum toxin, to a sympathetic ganglion which innervates a parathyroid hormone secreting parathyroid cell, thereby reducing a stimulatory effect upon parathyroid hormone secretion.

CROSS REFERENCE

This application is a divisional of Ser. No. 09/510,711, filed Feb. 22,2000 now abandoned.

BACKGROUND

The present invention relates to methods for treating parathyroiddisorders. In particular the present invention relates to methods fortreating parathyroid disorders by administration of a neurotoxin to apatient.

The adult human typically has four small parathyroid glands, eachweighing about 30 to 40 mg, located near the thyroid. The chief cells ofthe parathyroid glands can make and release parathyroid hormone (PTH),which functions to help maintain serum calcium homeostasis. Parathyroidhormone increases blood calcium level while calcitonin from the thyroidC cells acts to lower it.

Disorders of the parathyroid glands include hyperparathyroidism andhypoparathyroidism. Primary hyperparathyroidism is about twice asprevalent in females as it is in males, and this ratio increases withage. About 1 in 500 females over age of 40 and 1 in 2000 males over theage of 40 has primary hyperparathyroidism. In the United States about250,000 persons are afflicted with primary hyperparathyroidism.

Primary hyperparathyroidism exists when a disorder of parathyroid tissueitself, as the primary defect, results in the release into thecirculation of too much parathyroid hormone. Among the known causes ofprimary hyperparathyroidism are parathyroid adenoma, hyperplasia andcarcinoma. Secondary hyperparathyroidism is a reactive parathyroidhyperplasic phenomenon, which can accompany renal failure. Symptoms ofhyperparathyroidism can include nephrolithiasis, bone disease, pepticulcer, fatigue and hypertension.

Untreated hyperparathyroidism can result in the loss of considerableamounts of bone mass due to the hypercalcemia which arises from anexcessive level of circulating parathyroid hormone. Thus a high level ofparathyroid hormone causes osteoclastic bone reabsorption which can leadto multiple foci of bone destruction, osteitis fibrosa cystica or vonRecklinghausen's disease of bone.

Production of parathyroid hormone by the chief cells of the parathyroidglands is apparently regulated to a significant extent in the normalparathyroid by both free calcium concentration in extracellular fluidand by levels of 1,25 dihydroxyvitamin D (calcitriol). Parathyroidhormone is a single chain, 84 amino acid residue polypeptide which actsupon osteocytes and osteoclasts to increase the rate of release ofcalcium from bone into blood, apparently by stimulation of osteocyticosteolysis.

The treatment of choice for primary hyperparathyroidism is surgery toremove all or most of the hyperactive parathyroid tissue.Thallium-technetium subtraction scans, ultrasound, selective venoussampling, CT, MRI, and arteriography have been used to localize aparathyroid disorder. Unfortunately, it has been reported that in aboutone third of parathyroidectomies, surgery fails to cure thehyperparathyroidism because of surgical ineptness to remove theappropriate tissues. Furthermore, excessive removal of parathyroidglands tissue can cause tetany. Complications of parathyroidectomy caninclude hematoma, vocal cord paralysis, hypocalcemia, and persistenthypercalcemia. Thus, after parathyroidectomy 5% of patients havepermanent hypocalcemia, which therefore requires daily oralsupplementation or reimplantation of cryopreserved parathyroid tissue.

Significantly, while parathyroid adenoma can be treated by removal ofthe one abnormal parathyroid gland, removal of multiple parathyroidglands is typically required to treat parathyroid hyperplasia.Furthermore, the cause or causes of primary parathyroid hyperplasia areunknown.

Alternates to surgery for primary hyperparathyroidism include ethanolblock and embolization. Block by ethanol injection destroys theparathyroid gland or glands injected and can cause Horner's syndrome andvocal cord paralysis. Addditionally, embolization to the arterysupplying an abnormal parathyroid gland while sometimes successful toinfarct the parathyroid gland and normalize calcium levels, is adifficult procedure with a limited success rate.

Primary hypoparathyroidism due to deficient PTH secretion can cause alow serum calcium due to a lack of PTH mediated bone resorption andcalcium reabsorption by the kidneys. Symptoms of hypocalcemia includeneuromuscular irritability and tetany. Intravenous calcium is thetreatment of choice for primary hypoparathyroidism. Notably, PTHreplacement has also been used to treat primary hypoparathyroidism.Drawbacks to PTH replacement include lack of clinical experience, itmust be given by injection and it is expensive.

Parathyroid Innervation

With regard to parathyroid innervation, one view is that the nerves tothe parathyroids are only vasomotor, not secretomotor in nature, andthat parathyroid activity is controlled solely by variation in bloodcalcium level. Thus, a rise in blood calcium level inhibits PTH release,while a fall in blood calcium level stimulates PTH release.

Significantly it has been reported that parasympathetic influencesinhibit parathyroid hormone secretion, that cholinergic agonistsdecrease serum PTH and that this effect is blocked by atropine. See e.g.J. Auto Nerv Syst 1994;48:4-553, Metabolism 1985;34(7):612-615 andBrazilian J Med Biol Res 1994;27:573-599.

Additionally, the close anatomic association of the thyroid andparathyroid glands makes it reasonable to assume that the parathyroidsare innervated in a manner similar to the thyroid. The two upperparathyroid glands are located adjacent to the posterior surface of theupper or, middle part of the thyroid lobe, often just anterior to therecurrent laryngeal nerve as it enters the larynx. The two lowerparathyroid glands are usually found on the lateral or posteriorsurfaces of the lower part of the thyroid gland or within severalcentimeters of the lower thyroid pole within the thymic tongue.

It is known that the thyroid gland receives innervation from both thesympathetic and parasympathetic divisions of the autonomic nervoussystem. The sympathetic fibers arise from the cervical ganglia and enterwith blood vessels, whereas the parasympathetic fibers are derived fromthe vagus and reach the gland via branches of the laryngeal nerves. Thethyroid gland's relation to the recurrent laryngeal nerves and to theexternal branch of the superior laryngeal nerves is of major surgicalsignificance, since damage to these nerves can lead to a disability ofphonation.

Sympathetic innervation of the thyroid cells has been reported to exerta stimulatory effect upon thyroid hormone release through adrenergicreceptors for norepinephrine on follicle cells. Endocrinology1979;105:7-9. Significantly, the human thyroid is also innervated bycholinergic, parasympathetic fibers. Cell Tiss Res 1978;195:367-370. Seealso Biol Signals 1994;3:15-25. And other mammalian species are known toalso have cholinergicly innervated thyroid cells. See e.g. Z. MikroskAnat Forsch Leipzig 1986;100:1,S, 34-38 (pig thyroid is cholinergiclyinnervated); Neuroendocrinology 1991;53:69-74 (rat thyroid ischolinergicly innervated); Endocrinology 1984;114:1266-1271 (dog thyroidis cholinergicly innervated);

Significantly, the consensus is that cholinergic, parasympatheticinfluence upon thyroid hormone secretion by thyroid follicle cells ininhibitory. Endocrinology 1979;105:7-9; Endocrinology1984;114:126-61271; Peptides 985;6:58-5589; Peptides 1987;8:893-897,and; Brazilian J Med Biol Res 994;27:573-599. The direct cholinergicinfluence upon the thyroid appears to be mediated by muscarinicacetylcholine receptors of thyroid follicle cells since the cholinergicinhibition is blocked by atropine. Endocrinology 1979;105:7.

Thus, one can conclude that, at least in some circumstances, thedeficient PTH secretion of primary hypoparathyroidism is influenced byinhibitory parasympathetic innervation of the parathyroids, whileprimary parathyroid hyperplasia is influenced by excessive sympatheticstimulation of the parathyroids.

Botulinum Toxin

The anaerobic, gram positive bacterium Clostridium botulinum produces apotent polypeptide neurotoxin, botulinum toxin, which causes aneuroparalytic illness in humans and animals referred to as botulism.The spores of Clostridium botulinum are found in soil and can grow inimproperly sterilized and sealed food containers of home basedcanneries, which are the cause of many of the cases of botulism. Theeffects of botulism typically appear 18 to 36 hours after eating thefoodstuffs infected with a Clostridium botulinum culture or spores. Thebotulinum toxin can apparently pass unattenuated through the lining ofthe gut and attack peripheral motor neurons. Symptoms of botulinum toxinintoxication can progress from difficulty walking, swallowing, andspeaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the most lethal natural biological agent knownto man. About 50 picograms of a commercially available botulinum toxintype A (purified neurotoxin complex)¹ is a LD₅₀ in mice (i.e. 1 unit).Interestingly, on a molar basis, botulinum toxin type A is about 1.8billion times more lethal than diphtheria, about 600 million times morelethal than sodium cyanide, about 30 million times more lethal thancobra toxin and about 12 million times more lethal than cholera. Singh,Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) ofNatural Toxins II, edited by B. R. Singh et al., Plenum Press, New York(1996) (where the stated LD₅₀ of botulinum toxin type A of 0.3 ng equals1 U is corrected for the fact that about 0.05 ng of BOTOX® equals 1unit). One unit (U) of botulinum toxin is defined as the LD₅₀ uponintraperitoneal injection into female Swiss Webster mice weighing 18 to20 grams each.

¹Available from Allergan, Inc., of Irvine, Calif. under the tradenameBOTOX® in 100 unit vials. One unit of BOTOX® contains about 50 picogramsof botulinum toxin type A complex.

Seven immunologically distinct botulinum neurotoxins have beencharacterized, these being respectively botulinum neurotoxin serotypesA, B, C₁, D, E, F and G each of which is distinguished by neutralizationwith type-specific antibodies. The different serotypes of botulinumtoxin vary in the animal species that they affect and in the severityand duration of the paralysis they evoke. For example, it has beendetermined that botulinum toxin type A is 500 times more potent, asmeasured by the rate of paralysis produced in the rat, than is botulinumtoxin type B. Additionally, botulinum toxin type B has been determinedto be non-toxic in primates at a dose of 480 U/kg which is about 12times the primate LD₅₀ for botulinum toxin type A. Botulinum toxinapparently binds with high affinity to cholinergic motor neurons, istranslocated into the neuron and blocks the release of acetylcholine.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletalmuscles. Botulinum toxin type A has been approved by the U.S. Food andDrug Administration for the treatment of blepharospasm, strabismus andhemifacial spasm. Non-type A botulinum toxin serotypes apparently have alower potency and/or a shorter duration of activity as compared tobotulinum toxin type A. Clinical effects of peripheral intramuscularbotulinum toxin type A are usually seen within one week of injection.The typical duration of symptomatic relief from a single intramuscularinjection of botulinum toxin type A averages about three months.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and/orcleaving these proteins at different sites. For example, botulinum typesA and E both cleave the 25 kiloDalton (kD) synaptosomal associatedprotein (SNAP-25), but they target different amino acid sequences withinthis protein. Botulinum toxin types B, D, F and G act onvesicle-associated protein (VAMP, also called synaptobrevin), with eachserotype cleaving the protein at a different site. Finally, botulinumtoxin type C, has been shown to cleave both syntaxin and SNAP-25. Thesedifferences in mechanism of action may affect the relative potencyand/or duration of action of the various botulinum toxin serotypes.Significantly, it is known that the cytosol of pancreatic islet B cellscontains at least SNAP-25 (Biochem J 1;339 (pt 1): 159-65 (April 1999)),and synaptobrevin (Mov Disord 1995 May; 10(3): 376).

The molecular weight of the botulinum toxin protein molecule, for allseven of the known botulinum toxin serotypes, is about 150 kD.Interestingly, the botulinum toxins are released by Clostridialbacterium as complexes comprising the 150 kD botulinum toxin proteinmolecule along with associated non-toxin proteins. Thus, the botulinumtoxin type A complex can be produced by Clostridial bacterium as 900 kD,500 kD and 300 kD forms. Botulinum toxin types B and C₁ is apparentlyproduced as only a 500 kD complex. Botulinum toxin type D is produced asboth 300 kD and 500 kD complexes. Finally, botulinum toxin types E and Fare produced as only approximately 300 kD complexes. The complexes (i.e.molecular weight greater than about 150 kD) are believed to contain anon-toxin hemaglutinin protein and a non-toxin and non-toxicnonhemaglutinin protein. These two non-toxin proteins (which along withthe botulinum toxin molecule comprise the relevant neurotoxin complex)may act to provide stability against denaturation to the botulinum toxinmolecule and protection against digestive acids when toxin is ingested.Additionally, it is possible that the larger (greater than about 150 kDmolecular weight) botulinum toxin complexes may result in a slower rateof diffusion of the botulinum toxin away from a site of intramuscularinjection of a botulinum toxin complex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. Additionally, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP and glutamate.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and G cantherefore be recovered from bacterial cultures in predominantly theiractive form. In contrast, botulinum toxin serotypes C₁, D and E aresynthesized by. nonproteolytic strains and are therefore typicallyunactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains and therefore can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy.Additionally, it is known that botulinum toxin type B has, uponintramuscular injection, a shorter duration of activity and is also lesspotent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≧3×10⁷U/mg, an A₂₆₀/A₂₇₈ of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A, as set forth in Schantz, E. J., etal, Properties and use of Botulinum toxin and Other MicrobialNeurotoxins in Medicine, Microbiol Rev. 56: 80-99 (1992). Generally, thebotulinum toxin type A complex can be isolated and purified from ananaerobic fermentation by cultivating Clostridium botulinum type A in asuitable medium. The known process can also be used, upon separation outof the non-toxin proteins, to obtain pure botulinum toxins, such as forexample: purified botulinum toxin type A with an approximately 150 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater; purified botulinum toxin type B with an approximately 156 kDmolecular weight with a specific potency of 1-2×10⁸ LD₅₀ U/mg orgreater, and; purified botulinum toxin type F with an approximately 155kD molecular weight with a specific potency of 1-2×10⁷ LD₅₀ U/mg orgreater.

Already prepared and purified botulinum toxins and toxin complexessuitable for preparing pharmaceutical formulations can be obtained fromList Biological Laboratories, Inc., Campbell, Calif.; the Centre forApplied Microbiology and Research, Porton Down, U.K.; Wako (Osaka,Japan), as well as from Sigma Chemicals of St Louis, Mo.

Pure botulinum toxin is so labile that it is generally not used toprepare a pharmaceutical composition. Furthermore. the botulinum toxincomplexes, such a the toxin type A complex are also extremelysusceptible to denaturation due to surface denaturation, heat, andalkaline conditions. Inactivated toxin forms toxoid proteins which maybe immunogenic. The resulting antibodies can render a patient refractoryto toxin injection.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) is dependant, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of the toxincomplex obtained by the known culturing, fermentation and purificationto the much, much lower toxin concentrations used for pharmaceuticalcomposition formulation results in rapid detoxification of the toxinunless a suitable stabilizing agent is present. Dilution of the toxinfrom milligram quantities to a solution containing nanograms permilliliter presents significant difficulties because of the rapid lossof specific toxicity upon such great dilution. Since the toxin may beused months or years after the toxin containing pharmaceuticalcomposition is formulated, the toxin must be stabilized with astabilizing agent. The only successful stabilizing agent for thispurpose has been the animal derived proteins albumin and gelatin. And asindicated, the presence of animal derived proteins in the finalformulation presents potential problems in that certain stable viruses,prions or other infectious or pathogenic compounds carried through fromdonors can contaminate the toxin.

Furthermore, any one of the harsh pH, temperature and concentrationrange conditions required to lyophilize (freeze-dry) or vacuum dry abotulinum toxin containing pharmaceutical composition into a toxinshipping and storage format (ready for use or reconstitution by aphysician) can detoxify the toxin. Thus, animal derived or donor poolproteins such as gelatin and serum albumin have been used with somesuccess to stabilize botulinum toxin.

A commercially available botulinum toxin containing pharmaceuticalcomposition is sold under the trademark BOTOX® (available from Allergan,Inc., of Irvine, Calif.). BOTOX® consists of a purified botulinum toxintype A complex, albumin and sodium chloride packaged in sterile,vacuum-dried form. The botulinum toxin type A is made from a culture ofthe Hall strain of Clostridium botulinum grown in a medium containingN-Z amine and yeast extract. The botulinum toxin type A complex ispurified from the culture solution by a series of acid precipitations toa crystalline complex consisting of the active high molecular weighttoxin protein and an associated hemagglutinin protein. The crystallinecomplex is re-dissolved in a solution containing saline and albumin andsterile filtered (0.2 microns) prior to vacuum-drying. BOTOX® can bereconstituted with sterile, non-preserved saline prior to intramuscularinjection. Each vial of BOTOX® contains about 100 units (U) ofClostridium botulinum toxin type A purified neurotoxin complex, 0.5milligrams of human serum albumin and 0.9 milligrams of sodium chloridein a sterile, vacuum-dried form without a preservative.

To reconstitute vacuum-dried BOTOX® sterile normal saline without apreservative; 0.9% Sodium Chloride Injection is used by drawing up theproper amount of diluent in the appropriate size syringe. Since BOTOX®is denatured by bubbling or similar violent agitation, the diluent isgently injected into the vial. BOTOX® should be administered within fourhours after reconstitution. During this time period, reconstitutedBOTOX® is stored in a refrigerator (2° to 8° C.). Reconstituted BOTOX®is clear, colorless and free of particulate matter. The vacuum-driedproduct is stored in a freezer at or below −5° C. BOTOX® is administeredwithin four hours after the vial is removed from the freezer andreconstituted. During these four hours, reconstituted BOTOX® can bestored in a refrigerator (20 to 8° C.). Reconstituted BOTOX® is clear,colorless and free of particulate matter.

It has been reported that botulinum toxin type A has been used inclinical settings as follows:

(1) about 75-125 units of BOTOX® per intramuscular injection (multiplemuscles) to treat cervical dystonia;

(2) 5-10 units of BOTOX® per intramuscular injection to treat glabellarlines (brow furrows) (5 units injected intramuscularly into the procerusmuscle and 10 units injected intramuscularly into each corrugatorsupercilii muscle);

(3) about 30-80 units of BOTOX® to treat constipation by intrasphincterinjection of the puborectalis muscle;

(4) about 1-5 units per muscle of intramuscularly injected BOTOX® totreat blepharospasm by injecting the lateral pre-tarsal orbicularisoculi muscle of the upper lid and the lateral pre-tarsal orbicularisoculi of the lower lid.

(5) to treat strabismus, extraocular muscles have been injectedintramuscularly with between about 1-5 units of BOTOX®, the amountinjected varying based upon both the size of the muscle to be injectedand the extent of muscle paralysis desired (i.e. amount of dioptercorrection desired).

(6) to treat upper limb spasticity following stroke by intramuscularinjections of BOTOX® into five different upper limb flexor muscles, asfollows:

(a) flexor digitorum profundus: 7.5 U to 30 U

(b) flexor digitorum sublimus: 7.5 U to 30 U

(c) flexor carpi ulnaris: 10 U to 40 U

(d) flexor carpi radialis: 15 U to 60 U

(e) biceps brachii: 50 U to 200 U. Each of the five indicated muscleshas been injected at the same treatment session, so that the patientreceives from 90 U to 360 U of upper limb flexor muscle BOTOX® byintramuscular injection at each treatment session.

(7) to treat migraine, pericranial injected (injected symmetrically intoglabellar, frontalis and temporalis muscles) injection of 25 U of BOTOX®has showed significant benefit as a prophylactic treatment of migrainecompared to vehicle as measured by decreased measures of migrainefrequency, maximal severity, associated vomiting and acute medicationuse over the three month period following the 25 U injection.

The success of botulinum toxin type A to treat a variety of clinicalconditions has led to interest in other botulinum toxin serotypes. Astudy of two commercially available botulinum type A preparations(BOTOX® and Dysport®) and preparations of botulinum toxins type B and F(both obtained from Wako Chemicals, Japan) has been carried out todetermine local muscle weakening efficacy, safety and antigenicpotential. Botulinum toxin preparations were injected into the head ofthe right gastrocnemius muscle (0.5 to 200.0 units/kg) and muscleweakness was assessed using the mouse digit abduction scoring assay(DAS). ED₅₀ values were calculated from dose response curves. Additionalmice were given intramuscular injections to determine LD₅₀ doses. Thetherapeutic index was calculated as LD₅₀/ED₅₀. Separate groups of micereceived hind limb injections of BOTOX® (5.0 to 10.0 units/kg) orbotulinum toxin type B (50.0 to 400.0 units/kg), and were tested formuscle weakness and increased water consumption, the later being aputative model for dry mouth. Antigenic potential was assessed bymonthly intramuscular injections in rabbits (1.5 or 6.5 ng/kg forbotulinum toxin type B or 0.15 ng/kg for BOTOX®). Peak muscle weaknessand duration were dose related for all serotypes. DAS ED₅₀ values(units/kg) were as follows: BOTOX®: 6.7, Dysport®: 24.7, botulinum toxintype B: 27.0 to 244.0, botulinum toxin type F: 4.3. BOTOX® had a longerduration of action than botulinum toxin type B or botulinum toxin typeF. Therapeutic index values were as follows: BOTOX®: 10.5, Dysport®:6.3, botulinum toxin type B: 3.2. Water consumption was greater in miceinjected with botulinum toxin type B than with BOTOX®, althoughbotulinum toxin type B was less effective at weakening muscles. Afterfour months of injections 2 of 4 (where treated with 1.5 ng/kg) and 4 of4 (where treated with 6.5 ng/kg) rabbits developed antibodies againstbotulinum toxin type B. In a separate study, 0 of 9 BOTOX® treatedrabbits demonstrated antibodies against botulinum toxin type A. DASresults indicate relative peak potencies of botulinum toxin type A beingequal to botulinum toxin type F, and botulinum toxin type F beinggreater than botulinum toxin type B. With regard to duration of effect,botulinum toxin type A was greater than botulinum toxin type B, andbotulinum toxin type B duration of effect was greater than botulinumtoxin type F. As shown by the therapeutic index values, the twocommercial preparations of botulinum toxin type A (BOTOX® and Dysport®)are different. The increased water consumption behavior observedfollowing hind limb injection of botulinum toxin type B indicates thatclinically significant amounts of this serotype entered the murinesystemic circulation. The results also indicate that in order to achieveefficacy comparable to botulinum toxin type A, it is necessary toincrease doses of the other serotypes examined. Increased dosage cancomprise safety. Furthermore, in rabbits, type B was more antigenic thanwas BOTOX®, possibly because of the higher protein load injected toachieve an effective dose of botulinum toxin type B. Eur J Neurol 1999Nov;6(Suppl 4):S3-S10.

Acetylcholine

Typically only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system. Theneurotransmitter acetylcholine is secreted by neurons in many areas ofthe brain, but specifically by the large pyramidal cells of the motorcortex, by several different neurons in the basal ganglia, by the motorneurons that innervate the skeletal muscles, by the preganglionicneurons of the autonomic nervous system (both sympathetic andparasympathetic), by the postganglionic neurons of the parasympatheticnervous system, and by some of the postganglionic neurons of thesympathetic nervous system. Essentially, only the postganglionicsympathetic nerve fibers to the sweat glands, the piloerector musclesand a few blood vessels are cholinergic as most of the postganglionicneurons of the sympathetic nervous system secret the neurotransmitternorepinephine. In most instances acetylcholine has an excitatory effect.However, acetylcholine is known to have inhibitory effects at some ofthe peripheral parasympathetic nerve endings, such as inhibition ofheart rate by the vagal nerve.

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Since,the preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic neurons of the parasympathetic nervoussystem, as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the synapses between the preganglionic and postganglionicneurons of both the sympathetic and parasympathetic. The nicotinicreceptors are also present in many membranes of skeletal muscle fibersat the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as, adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and parathyroid hormone, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,permeabilized (as by electroporation) or by direct injection of thetoxin into the denervated cell. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

What is needed therefore is an effective, long lasting, non-surgicalresection, non-radiotherapy, non-systemic drug administration,therapeutic drug and method for treating parathyroid disorders.

SUMMARY

The present invention meets this need and provides an effective,non-surgical resection, long term, non-radiotherapy, non-systemic drugadministration, therapeutic method for treating parathyroid disorders.

The drug within the scope of this invention for treating parathyroiddisorders is a neurotoxin. Significantly, the same neurotoxin can beused to treat hyperparathyroidism, hypoparathyroidism, hypocalcemia andhypercalcemia depending upon factors such as the site of localadministration of the neurotoxin and the amount of neurotoxin to beadministered.

As used herein “local administration” means direct injection of aneurotoxin into a parathyroid gland or into a sympathetic ganglion whichinnervates a parathyroid PTH secretory cell. Systemic routes ofadministration, such as oral and intravenous routes of administration,are excluded from the scope of “local administration” of a neurotoxin.

A method for treating a parathyroid disorder according to the presentinvention can be carried out by administration of a therapeuticallyeffective amount of a neurotoxin to a patient, thereby treating theparathyroid disorder. The neurotoxin can administered to a parathyroidgland of the patient when the parathyroid disorder to be treated ishypoparathyroidism. Alternately, the neurotoxin can be administered to asympathetic ganglion which innervates a parathyroid PTH secreting cellwhen the parathyroid disorder to be treated is hyperparathyroidism.

A detailed method for treating a parathyroid disorder according to thepresent invention can comprise the step of administration of atherapeutically effective amount of a botulinum toxin to a patient.Thus, a method for treating hypoparathyroidism according to the presentinvention can comprise the step of local administration to a parathyroidgland (i.e. to one or more of the four normally present parathyroidglands) of a therapeutically effective amount of a botulinum toxin,thereby increasing a deficient parathyroid hormone secretion from aparathyroid cell which is capable of secreting parathyroid hormone, andeffectively treating the hypoparathyroidism. Furthermore, a methodwithin the scope of the present invention for treatinghyperparathyroidism, can comprise the step of local administration to asympathetic ganglion which innervates a parathyroid PTH secreting cellof a parathyroid gland of a therapeutically effective amount of abotulinum toxin, thereby reducing an excessive parathyroid hormonesecretion from the parathyroid cell and hence effectively treating thehyperparathyroidism.

The neurotoxin can be administered in an amount of between about 10⁻³U/kg and about 35 U/kg. 35 U/kg is an upper limit because it approachesa lethal dose of certain neurotoxins, such as botulinum toxin type A.Other botulinum toxins, such as botulinum toxin type B, can be safelyadministered at several orders of magnitude higher dosage. Preferably,the neurotoxin is administered in an amount of between about 10⁻² U/kgand about 25 U/kg. More preferably, the neurotoxin is administered in anamount of between about 10^(″1) U/kg and about 15 U/kg. Most preferably,the neurotoxin is administered in an amount of between about 1 U/kg andabout 10 U/kg. In many instances, an administration of from about 0.1units to about 300 units of a neurotoxin, such as a botulinum toxin typeA, provides effective and long lasting therapeutic relief. Morepreferably, from about 0.1 unit to about 100 units of a neurotoxin, suchas a botulinum toxin type A, can be used and most preferably, from about0.1 unit to about 50 units of a neurotoxin, such as a botulinum toxintype A, can be locally administered into a target tissue such as thethyroid or a sympathetic ganglion with efficacious results. In aparticularly preferred embodiment of the present invention, aparathyroid gland or a sympathetic ganglion which innervates aparathyroid gland, can be locally administered with from about 1 unit toabout 20 units of a neurotoxin (such as botulinum toxin type A) toachieve therapeutically effective results.

The neurotoxin can be made by a Clostridial bacterium, such as by aClostridium botulinum, Clostridium butyricum, Clostridium beratti orClostridium tetani bacterium. Additionally, the neurotoxin can be amodified neurotoxin, that is a neurotoxin that has at least one of itsamino acids deleted, modified or replaced, as compared to the native orwild type neurotoxin. Furthermore, the neurotoxin can be a recombinantproduced neurotoxin or a derivative or fragment thereof.

The neurotoxin can be a botulinum toxin, such as one of the botulinumtoxin serotypes A, B, C₁D, E, F or G. Preferably, the neurotoxin isbotulinum toxin type A and the neurotoxin is locally administered bydirect injection of the neurotoxin into a parathyroid gland or into asympathetic ganglion which innervates the parathyroid gland.

A detailed embodiment of a method within the scope of the presentinvention for treating a parathyroid disorder can comprise the step ofinjecting a therapeutically effective amount of a botulinum toxin into aparathyroid gland of a human patient, thereby increasing a parathyroidhormone (PTH) secretion from a parathyroid hormone secreting parathyroidcell of the parathyroid gland and treating a parathyroid disorder.

Another detailed embodiment of a method within the scope of the presentinvention for treating a parathyroid disorder of a human patient cancomprise the step of local administration to a cholinergic influencedparathyroid PTH secreting cell of a parathyroid gland of a human patientof a therapeutically effective amount of botulinum toxin type A, therebyincreasing a cholinergic influenced deficient parathyroid hormonesecretion from the parathyroid cell of the parathyroid gland andtreating the parathyroid disorder.

Another method within the scope of the present invention is a method fortreating a parathyroid disorder by administration of a neurotoxin to asympathetic nervous system of a patient. In this method the neurotoxinis locally administered to a sympathetic ganglion which innervates aparathyroid hormone secreting parathyroid cell and the parathyroiddisorder is hyperparathyroidism.

A detailed embodiment of method within the scope of the presentinvention for treating a parathyroid disorder of a human patient cancomprise the step of in vivo, local administration to a sympatheticganglion, which innervates a parathyroid hormone secreting prothyroidcell of a parathyroid gland of a patient, of a therapeutically effectiveamount of a botulinum toxin, thereby decreasing an excessive parathyroidhormone secretion from the parathyroid cell of the parathyroid gland andtreating hyperparathyroidism.

A detailed embodiment of the present invention is a method for treatinga parathyroid disorder by injecting a therapeutically effective amountof a botulinum toxin into a parathyroid gland of a human patient,thereby increasing a secretion of a parathyroid hormone from aparathyroid cell and treating the parathyroid disorder. Preferably, thesecretion treated is a cholinergic influenced secretion and thebotulinum toxin used is botulinum toxin type A, although the botulinumtoxin can selected from the group consisting of botulinum toxin types A,B, C (i.e. C₁), D, E, F and G.

My invention also includes within its scope, a method for treatinghypocalcemia, the method comprising the step of local administration toa parathyroid hormone secreting parathyroid cell of a parathyroid glandof a therapeutically effective amount of a botulinum toxin, therebyincreasing a deficient parathyroid hormone secretion from theparathyroid cell and treating hypocalcemia. Additionally, my inventionalso includes within its scope a method for treating hypercalcemia, themethod comprising the step of local administration to a sympatheticganglion which innervates a parathyroid hormone secreting parathyroidcell of a parathyroid gland of a therapeutically effective amount of abotulinum toxin, thereby decreasing an excessive parathyroid hormonesecretion from the parathyroid cell of the parathyroid gland andtreating hypercalcemia.

DESCRIPTION

The present invention is based upon the discovery that a parathyroiddisorder can be treated by in vivo administration of a neurotoxin to apatient. Thus administration of a neurotoxin to a parathyroid gland of apatient can remove an inhibitory cholinergic effect upon parathyroidhormone secretion by the parathyroid gland, thereby providing aneffective treatment for hypoparathyroidism and/or hypocalcemia.Additionally, administration of a neurotoxin to a sympathetic ganglionwhich innervates a parathyroid hormone secretory cell of a parathyroidgland can remove a stimulatory adrenergic effect upon parathyroidhormone secretion, thereby providing an effective treatment forhyperparathyroidism and/or hypercalcemia.

Thus, parathyroid disorders can be treated, according to the presentinvention, by the alternative therapies of (a) local administration of aneurotoxin to one or more of the parathyroid glands, or; (b) localadministration of a neurotoxin to a parathyroid gland innervatingsympathetic ganglion of a patient, thereby resulting in, respectively,an increase of a secretion from a parathyroid cell, or a decrease in asecretion from a parathyroid chief (PTH secretory capable) cell

I have discovered that a particular neurotoxin, botulinum toxin, can beused with dramatic ameliorative effect to treat a parathyroid disorder,thereby significantly superseding thereby current therapeutic regimens,such as surgical removal of parathyroid gland tissue to treathyperparathyroidism and calcium supplementation to treathypoparathyroidism.

Significantly, a single local administration of a neurotoxin, such as abotulinum toxin to one or more of the parathyroid glands, according tothe present invention, can increase parathyroid hormone secretion andthereby treat symptoms of hypoparathyroidism. I have also discoveredthat a single local administration of a neurotoxin, such as a botulinumtoxin to one or more of the sympathetic ganglia which innervate aparathyroid gland, according to the present invention, can reduceparathyroid hormone secretion and thereby treat symptoms ofhyperparathyroidism. In either case, the symptoms of the parathyroiddisorder can be alleviated for at least about from 2 months to about 6months per neurotoxin administration. Notably, it has been reported thatglandular tissue treated by a botulinum toxin can show a reducedsecretory activity for as long as 27 months post injection of the toxin.Laryngoscope 1999; 109:1344-1346, Laryngoscope 1998;108:381-384. Myinvention also includes within its scope the use of an implantedsustained release neurotoxin complex so as to provide therapeutic relieffrom a chronic parathyroid disorder. Thus, the neurotoxin can beimbedded within, absorbed, or carried by a suitable polymer matrix whichcan be implanted or embedded in or on a parathyroid gland or sympatheticganglion so as to provide a year or more of delayed and controlledrelease of the neurotoxin to the desired target tissue.

The hypoparathyroidism treatable by the present invention is primaryhypoparathyroidism. Secondary hypoparathyroidism is not treatable by thepresent invention, because the present invention is based upon atherapeutic, local administration of a neurotoxin to one or more of theparathyroid glands and/or to a sympathetic ganglion which innervates theone or more of the parathyroid glands. Additionally the primaryhypoparathyroidism treatable by the present invention ishypoparathyroidism which has as a causative factor the inhibitoryactivity upon parathyroid hormone secretion of cholinergic,parasympathetic innervation of the parathyroid.

Similarly, the hyperparathyroidism treatable by the present invention isprimary hyperparathyroidism. Secondary hyperparathyroidism is nottreatable by the present invention, because the present invention isbased upon a therapeutic, local administration of a neurotoxin to one ormore of the parathyroid glands and/or to a sympathetic ganglion whichinnervates the one or more of the parathyroid glands. Additionally theprimary hyperparathyroidism treatable by the present invention ishyperparathyroidism which has as a causative factor the stimulatoryactivity upon parathyroid hormone secretion of sympathetic innervationof the parathyroid.

Notably, hypoparathyroidism resulting from a combination of factors,including inhibitory parasympathetic activity, is treatable by a methodwithin the scope of the present invention. Similarly,hyperparathyroidism resulting from a combination of factors, includingstimulatory sympathetic activity, is treatable by a method within thescope of the present invention.

Local Administration of a Neurotoxin to the Parathyroid

A preferred embodiment of the present invention is to inject aparathyroid gland of a patient with from 0.1 to 100 units, morepreferably from 1 to 50 units, and most preferably from 1 to 10 units ofa neurotoxin (such as a botulinum toxin type A), to thereby cause anincrease of parathyroid hormone secretion. The present invention alsoincludes within its scope treatment of a parathyroid disorder due tohyperplasic, hypertonic or hypertrophic parathyroid chief cells. Aparathyroid disorder can be effectively treated by local administrationof a neurotoxin, such as for example 0.1 to 100 units of botulinum toxintype A, to cholinergic, postganglionic, parasympathetic neurons whichinnervate the dysfunctional, parathyroid cells. Without wishing to bebound by theory, the botulinum toxin is believed to act to increaseparathyroid hormone secretion by inhibiting release of acetylcholineneurotransmitter from cholinergic, postganglionic parasympathetic fiberswhich provide inhibitory innervation of parathyroid PTH secreting cells.

A neurotoxin, such as a botulinum toxin, locally administered in vivo tothe parathyroid to thereby remove an inhibitory effect upon a secretoryactivity of a parathyroid hormone secreting parathyroid cell. Theparathyroid hormone secreting parathyroid cell is cholinergicallyinnervated such that the proteolytic light chain of the toxin isinternalized by a cholinergic neuron which inhibitoraly influences asecretory activity of the parathyroid hormone secreting parathyroidcell.

Thus, cholinergically innervated parathyroid cells can be treated bylocal administration of a neurotoxin, such as a botulinum toxin. Bylocal administration, of a parathyroid gland, it is meant that theneurotoxin is administered directly to or to the immediate vicinity ofthe parathyroid tissue to be treated.

The specific dosage appropriate for administration is readily determinedby one of ordinary skill in the art according to the factor discussedabove. The dosage can also depend upon the size of the parathyroidtissue mass to be treated or denervated, and the commercial preparationof the toxin. Additionally, the estimates for appropriate dosages inhumans can be extrapolated from determinations of the amounts ofbotulinum required for effective denervation of other tissues. Thus, theamount of botulinum A to be injected is proportional to the mass andlevel of activity of the thyroid tissue to be treated. Generally,between about 0.01 and 35 units per kg of patient weight of a botulinumtoxin, such as botulinum toxin type A, can be administered toeffectively accomplish a toxin induced parathyroid tissue secretion downregulation upon administration of the neurotoxin into the thyroid. Lessthan about 0.01 U/kg of a botulinum toxin does not have a significanttherapeutic effect upon the secretory activity of a parathyroid cell,while more than about 35 U/kg of a botulinum toxin approaches a toxicdose the neurotoxin. Careful placement of the injection needle and a lowvolume of neurotoxin used prevents significant amounts of botulinumtoxin from appearing systemically. A more preferred dose range is fromabout 0.01 U/kg to about 25 U/kg of a botulinum toxin, such as thatformulated as BOTOX®. The actual amount of U/kg of a botulinum toxin tobe administered depends upon factors such as the extent (mass) and levelof activity of the thyroid tissue to be treated and the administrationroute chosen. Botulinum toxin type A is a preferred botulinum toxinserotype for use in the methods of the present invention.

Diagnostic aids to determine hyper or hypoparathyroidism, bylocalization of the dysfunctional parathyroid tissue, includethallium-201/technetium-99m subtraction scintigraphy, computedtomography, ultrasonography, and magnetic resonance. Localizationstudies help by suggesting the most direct approach to the abnormalparathyroid tissue including whether surgery begins with a neck ormediastinal exploration. Particular scintigraphic techniques used tolocalize hyper or hypoplasic parathyroid glands, have included acombination of radiotracers including thallium-201 chloride ortechnetium-99m sestamibi and technetium-99m-sodium pertechnetate oriodine-123 sodium iodide. The processing of digital images obtainedincludes background correction and subtraction of the parathyroid imagefrom the composite parathyroid and parathyroid image. These methodsexploit the different biological behavior and tissue distribution of thetwo tracers, namely the parathyroid specific pertechnetate or iodineuptake relative to the more diffuse perfusion dependent distribution ofthallium or sestamibi.

It has been reported that the neuronal selectivity of clostridialneurotoxins is a result of a very selective binding and cell entrymechanism. Although a site of action of botulinum toxin is theneuromuscular junction, where the toxin binds rapidly and prevents therelease of acetylcholine from cholinergic neurons, it is known thatclostridial neurotoxins are able to enter certain neurosecretory cells(for example PC12 cells) via a low affinity receptor if highconcentrations of the neurotoxin are incubated with the cells forprolonged periods. This process appears to use a pathway via a receptorwhich is distinct from the highly specific and high affinity receptorpresent at the neuromuscular junction. Additionally, it has beenreported that certain clostridial toxins have effects on phagocytecells, such as macrophages, where entry into the cell is presumed to bevia the specific phagocytic activity of these cells. Furthermore,incubation of certain adipocytes (i.e. fat cells) with botulinum toxintype A has been reported to inhibit glucose uptake by the adipocytes.The mechanism of the glucose uptake inhibition is apparently due totoxin inhibition of plasma membrane fusion or docking of cytosolic,recyclable membrane vesicles (RMVs), the RMVs containing glucosetransporter proteins. PCT publication WO 94/21300.

Thus, while it is known that the botulinum toxins have a known bindingaffinity for cholinergic, pre-synaptic, peripheral motor neurons, it hasbeen reported that botulinum toxins can also bind to and translocateinto a variety of non-neuronal secretory cells, where the toxin thenacts, in the known manner, as an endoprotease upon its respectivesecretory vessel-membrane docking protein. Because of the relativelylower affinity of the botulinum toxins for secretory cells, such asparathyroid cells, as compared to the affinity of the botulinum toxinfor the cholinergic neurons which innervate parathyroid chief cells, thebotulinum toxin can be injected into secretory or glandular tissues toprovide a high local concentration of the toxin, thereby facilitatingeffect of the toxin upon both cholinergic neuron and directly uponparathyroid secretory cell. Thus, the present invention is applicable tothe treatment of parathyroid disorders in circumstances where the targetparathyroid secretory cells have little or no cholinergic innervation.Local administration of a neurotoxin to the parathyroids at a high doselevel is carried out to treat primary hyperparathyroidism.

Preferably, a neurotoxin used to practice a method within the scope ofthe present invention is a botulinum toxin, such as one of the serotypeA, B, C, D, E, F or G botulinum toxins. Preferably, the botulinum toxinused is botulinum toxin type A, because of its high potency in humans,ready availability, and known safe and efficacious use for the treatmentof skeletal muscle and smooth muscle disorders when locally administeredby intramuscular injection.

The present invention includes within its scope the use of anyneurotoxin which has a long duration therapeutic effect when locallyapplied to treat a parathyroid cell disorder of a patient. For example,neurotoxins made by any of the species of the toxin producingClostridium bacteria, such as Clostridium botulinum, Clostridiumbutyricum, and Clostridium beratti can be used or adapted for use in themethods of the present invention. Additionally, all of the botulinumserotypes A, B, C, D, E , F and G can be advantageously used in thepractice of the present invention, although type A is the most preferredserotype, as explained above. Practice of the present invention canprovide effective relief of a parathyroid disorder for from 2-27 monthsor longer in humans from a single injection of the neurotoxin.

Botulinum toxin is believed to be able to block the release of anyvesicle mediated exocytosis from any secretory (i.e. neuronal,glandular, secretory, chromaffin) cell type, as long as the light chainof the botulinum toxin is translocated into the intracellular medium.For example, the intracellular protein SNAP-25 is widely distributed inboth neuronal and non-neuronal secretory cells and botulinum toxin typeA is an endopeptidase for which the specific substrate is SNAP-25. Thus,while cholinergic neurons have a high affinity acceptor for thebotulinum and tetanus toxins (and are therefore more sensitive thanother neurons and other cells to the inhibition of vesicle mediatedexocytosis of secretory compounds), as the toxin concentration israised, non-cholinergic sympathetic neurons, chromaffin cells and othercell types can take up a botulinum toxin and show reduced exocytosis.

Hence, by practice of the present disclosed invention secretoryparathyroid cells with little or no cholinergic innervation can betreated by use of an appropriately higher concentration of a botulinumtoxin to bring about therapeutic relief from a parathyroid disorder suchas primary hyperparathyroidism. Local administration to a parathyroidgland of a lower dose neurotoxin (i.e. from about 1 unit to about 20units of botulinum toxin type A per parathyroid gland) can be used toteat hypoparathyroidism, as previously set forth.

Local Administration of a Neurotoxin to a Sympathetic Ganglion

Significantly, a method within the scope of the present invention forreducing an excessive parathyroid hormone secretion from a parathyroidgland comprises the step of local administration of a neurotoxin to thesympathetic nervous system. Sympathetic innervation of the parathyroidis know to exist. Thus, sympathetic nerve fibers can inhibit parathyroidhormone secretion by acting via adrenergic receptors on parathyroidhormone secreting parathyroid cells. A method within the scope of thepresent invention can therefore be carried out by local administrationof a neurotoxin to a preganglionic sympathetic (i.e. cholinergic) neuronwhich innervates a parathyroid cell. The cholinergic, preganglionic,sympathetic neuron synapses with adrenergic, post-ganglionic,sympathetic fibers, and these later sympathetic neurons have astimulatory effect upon parathyroid hormone secretion by parathyroidgland chief cells. Preferably, the sympathetic ganglion to which aneurotoxin is administered, according to the preset invention, is acervical ganglion.

Cervical ganglion block according to the present invention can becarried out in the same manner as a celiac plexus block. Thus, theneurolytic celiac plexus block is a known procedure for treatingintractable pain resulting from upper abdominal viscera cancer. RegAnest Pain Med 1998; 23(1):37-48. Thus, it is known to inject the celiacplexus with ethanol or phenol to provide relief from the pain which canresult from pancreatic cancer or from pancreatitis. AJG1999;94(4):872-874. Hence, an antinociceptive injection of the cervicalganglia can be carried out as by either a percutaneous procedure or asan open (intraoperative) injection. The percutaneous (closed) procedurecan be carried out using an anterior approach using a very thin needle(22 Gauge). Cervical ganglion block is preferably carried out withcomputed tomography (CT) (as opposed to fluoroscopic) needle guidance,using a single thin needle.

Furthermore, a method within the scope of the present invention canprovide improved patient function. “Improved patient function” can bedefined as an improvement measured by factors such as a reduced pain,reduced time spent in bed, increased ambulation, healthier attitude,more varied lifestyle and/or healing permitted by normal muscle tone.Improved patient function is synonymous with an improved quality of life(QOL). QOL can be assesses using, for example, the known SF-12 or SF-36health survey scoring procedures. SF-36 assesses a patient's physicaland mental health in the eight domains of physical functioning, rolelimitations due to physical problems, social functioning, bodily pain,general mental health, role limitations due to emotional problems,vitality, and general health perceptions. Scores obtained can becompared to published values available for various general and patientpopulations.

As set forth above, I have discovered that a surprisingly effective andlong lasting therapeutic effect can be achieved by local administrationof a neurotoxin to a parathyroid or to a sympathetic ganglion whichinnervates a parathyroid hormone secreting parathyroid cell of a humanpatient. In its most preferred embodiment, the present invention ispracticed by direct injection into a parathyroid gland, or into asympathetic ganglion which innervates a parathyroid gland, of atherapeutically effective amount of botulinum toxin, such as botulinumtoxin type A. It has been reported that at the neuroglandular junction,the chemical denervation effect of a single injection of botulinumtoxin, such as botulinum toxin type A, has a duration of action of up to27 months.

The present invention includes within its scope: (a) neurotoxin complexas well as pure neurotoxin obtained or processed by bacterial culturing,toxin extraction, concentration, preservation, freeze drying and/orreconstitution and; (b) modified or recombinant neurotoxin, that isneurotoxin that has had one or more amino acids or amino acid sequencesdeliberately deleted, modified or replaced by known chemical/biochemicalamino acid modification procedures or by use of known hostcell/recombinant vector recombinant technologies, as well as derivativesor fragments of neurotoxins so made, and includes neurotoxins with oneor more attached targeting moieties for a cell surface receptor presenton a parathyroid hormone secreting parathyroid cell.

Botulinum toxins for use according to the present invention can bestored in lyophilized or vacuum dried form in containers under vacuumpressure. Prior to lyophilization the botulinum toxin can be combinedwith pharmaceutically acceptable excipients, stabilizers and/orcarriers, such as albumin. The lyophilized or vacuum dried material canbe reconstituted with saline or water.

The route of administration and amount of a neurotoxin (such as abotulinum toxin serotype A, B, C, D, E, F or G) administered accordingto the present invention for treating a parathyroid disorder can varywidely according to various patient variables including size, weight,age, disease severity, responsiveness to therapy, and solubility anddiffusion characteristics of the neurotoxin toxin chosen. Furthermore,the extent of the parathyroid or ganglionic tissue influenced isbelieved to be proportional to the volume of neurotoxin injected, whilethe quantity of the denervation is, for most dose ranges, believed to beproportional to the concentration of neurotoxin injected.

Methods for determining the appropriate route of administration anddosage are generally determined on a case by case basis by the attendingphysician. Such determinations are routine to one of ordinary skill inthe art (see for example, Harrison's Principles of Intemal Medicine(1998), edited by Anthony Fauci et al., 14^(th) edition, published byMcGraw Hill). For example, to treat a parathyroid disorder, a solutionof botulinum toxin type A complex can be endoscopically orintraperitoneally injected directly into the tissues of the parathyroid,thereby substantially avoiding entry of the toxin into the systemiccirculation.

EXAMPLES

The following examples provide those of ordinary skill in the art withspecific preferred methods within the scope of the present invention forcarrying out the present invention and are not intended to limit thescope of what the inventor regards as his invention. In each of thefollowing examples, the specific amount of a botulinum toxinadministered depends upon a variety of factors to be weighed andconsidered within the discretion of the attending physician and in eachof the examples insignificant amounts of botulinum toxin enter appearsystemically with no significant side effects. Units of botulinum toxininjected per kilogram (U/kg) below are per kg of total patient weight.For example, 3 U/kg for a 70 kg patient calls for an injection of 210units of the botulinum toxin.

Example 1 Intraoperative Administration of Neurotoxin

Intraoperative, local administration of a neurotoxin to a parathyroidcan be carried out as follows. The procedure can be performed undergeneral endotracheal anesthesia. The patient's neck can be extended byinflating a pillow or inserting a thyroid roll beneath the shoulders. Asymmetrical, low, collar incision can then be made in the line of anatural skin crease approximately 1 to 2 cm above the clavicle. Theincision can be carried through the skin, subcutaneous tissue, andplatysma muscle down to the dense cervical fascia that overlies thestrap muscles and anterior jugular veins. The upper flap can then beraised to a level cephalad to the cricoid cartilage. Care is taken toavoid cutting sensory nerves. A small lower flap is also elevated to thelevel of the manubrial notch. Performing dissection of the flaps in theplane between the platysma muscle and the fascia overlying the strapmuscles results in minimal bleeding. The cervical fascia is then incisedvertically in the midline.

Exposure of the parathyroid glands can generally be achieved byretracting the sternohyoid and sternothyroid muscles laterally Digitalor blunt dissection frees the parathyroids from the surrounding fascia.An exposed parathyroid gland can be directly injected with from 0.1 to50 units of a botulinum toxin, such as botulinum toxin type A. Careshould be taken to ensure that the parathyroid glands are not excised ordevascularized. Within one to seven days, parathyroid hormone secretionis substantially increased due to removal of cholinergic inhibition andthis effect persists for from about 2 to about 6 months.

Example 2 Local Administration of Neurotoxin to a Parathyroid

Local administration of a neurotoxin directly to or to the vicinity of aparathyroid gland can be accomplished by several methods. For example,by parathyroid endoscopy. An endoscope used for parathyroid therapy canbe modified to permit its use for direct injection of a neurotoxin, suchas a botulinum toxin directly into parathyroid tissue. See for exampleU.S. Pat. No. 5,674,205. Once appropriately located, a hollow needle tipcan be extended from the endoscope into parathyroid tissue and throughwhich needle the neurotoxin can be injected into the parathyroid tissueof one or more of the parathyroid glands.

Additionally, fine needle aspiration for parathyroid biopsy purposes isknown and can be used to inject a neurotoxin, rather than to aspirateparathyroid tissue. From 0.1 to 50 units of a botulinum toxin, such asbotulinum toxin type A can thereby be injected into one or more of theparathyroid glands. Within one to seven days, parathyroid hormonesecretion is substantially increased due to removal of cholinergicinhibition and this effect persists for from 2 to 6 months.

Example 3 Treatment of Hypoparathyroidism With Botulinum Toxin Type A

A 43 year old male is diagnosed with hypoparathyroidism. Between about0.1 U and about 50 U of a botulinum toxin type A preparation (forexample between about 0.1 units and about 50 units of BOTOX®) isinjected directly into one or more of the parathyroid glands, using oneof the techniques set forth in Examples 1 or 2 above. Within 1-7 daysthe symptoms of the hypoparathyroidism are alleviated and parathyroidhormone levels return to substantially normal levels. Alleviation of theparathyroid disorder persists for at least about 2 months to about 6months.

Example 4 Treatment of Hypoparathyroidism With Botulinum Toxin Type B

A 52 year old female is diagnosed with hypoparathyroidism. Between about20 units and about 1000 units of a botulinum type B preparation isinjected directly into one or more of the parathyroids, using one of thetechniques set forth in Examples 1 or 2 above. Within 1-7 days thesymptoms of the hypoparathyroidism are alleviated. Parathyroid hormonelevels return to substantially normal levels. Alleviation of theparathyroid disorder persists for at least about 2 months to about 6months.

Example 5 Treatment of Hypoparathyroidism With Botulinum Toxin Type C

A 58 year old female is diagnosed with hypoparathyroidism. Between about10⁻³ U/lkg and about 35 U/kg of a botulinum toxin type C preparation(for example between about 10 units and about 10,000 units of abotulinum type C preparation) is injected directly into one or more ofthe parathyroids, using one of the techniques set forth in Examples 1 or2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 6 Treatment of Hypoparathyroidism With Botulinum Toxin Type D

A 56 year old obese female is diagnosed with hypoparathyroidism. Betweenabout 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Dpreparation (for example between about 10 units and about 10,000 unitsof a botulinum type D preparation) is injected directly into one or moreof the parathyroids, using one of the techniques set forth in Examples 1or 2 above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 7 Treatment of Hypoparathyroidism With Botulinum Toxin Type E

A 61 year old female is diagnosed with hypoparathyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type E preparation (forexample between about 10 units and about 10,000 units of a botulinumtype E preparation) is injected directly into one or more of theparathyroids, using one of the techniques set forth in Examples 1 or 2above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 8 Treatment of Hypoparathyroidism With Botulinum Toxin Type F

A 52 year old male is diagnosed with hypoparathyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type F preparation (forexample between about 10 units and about 10,000 units of a botulinumtype F preparation) is injected directly into one or more of theparathyroids, using one of the techniques set forth in Examples 1 or 2above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 9 Treatment of Hypoparathyroidism With Botulinum Toxin Type G

A 59 year old female is diagnosed with hypoparathyroidism. Between about10⁻³ U/kg and about 35 U/kg of a botulinum toxin type G preparation (forexample between about 10 units and about 10,000 units of a botulinumtype G preparation) is injected directly into one or more of theparathyroids, using one of the techniques set forth in Examples 1 or 2above. Within 1-7 days the symptoms of the hypoparathyroidism arealleviated. Parathyroid hormone levels return to substantially normallevels. Alleviation of the parathyroid disorder persists for at leastabout 2 months to about 6 months.

Example 10 Treatment of Hyperparathyroidism With Botulinum Toxin Type A

A 27 year old female presents with symptoms of progressively worseningmyalgias over the last six months. She was otherwise asymptomatic and ingood health. Physical examination is unremarkable. Family history andsocial history are likewise noncontributory. A routine screening serumchemistry profile reveals a serum calcium of 12.0 mg/dl. Serum albumin,protein, magnesium, and chloride are all within normal limits. Aparathormone assay level of 211 picograms/ml (normal=10 to 55picograms/ml) is noted. Two subsequent calcium levels and parathormonelevels are persistently elevated and the diagnosis of primaryhyperparathyroidism is made. A trial with atropine reduces theparathyroid hormone level.

Between about 10⁻³ U/kg and about 35 U/kg of a botulinum toxin type Apreparation (for example between about 0.1 units and about 50 units ofBOTOX®) is injected directly into the cervical ganglia as follows. Apercutaneous procedure is carried out using an anterior approach withthe patient in a supine position using a very thin needle (22 Gauge)with computed tomography needle guidance to reach the cervical ganglia.Within 1-7 days the symptoms of the hyperparathyroidism are alleviated.Parathyroid hormone levels return to normal (are lowered). Alleviationof the parathyroid disorder persists for at least about 2 months toabout 6 months.

Example 11 Treatment of Hyperparathyroidism With Botulinum Toxin TypesB-G

A 62 year old female presents with symptomatic hypercalcemia, serumcalcium of 1-1.6 mg/dL above normal values, a creatinine clearancereduction greater than 30%, a 24-hr. urine calcium excretion >400 mg,and a bone mass <2S.D.'s below normal. A diagnosis of primaryhyperparathyroidism is made. Between about 10⁻³ U/kg and about 35 U/kgof a botulinum toxin type B, C₁, D, E, F or G preparation (for examplebetween about 10 units and about 10,000 units of a botulinum type B-Gpreparation) is injected directly into the cervical ganglia as follows.A percutaneous procedure is carred out using an anterior approach withthe patient in a supine position using a very thin needle (22 Gauge)with computed tomography needle guidance to reach the cervical ganglia.Within 1-7 days the symptoms of the hyperparathyroidism are alleviatedand parathyroid hormone levels return to substantially normal levels.Alleviation of the hyperparathyroidism persists for at least about 2months to about 6 months.

Example 12 Treatment of Calcium Metabolism Disorders With BotulinumToxin Types A-G

A 28 year old female is diagnosed with hypocalcemia. Between about 10⁻³U/kg and about 35 U/kg of a botulinum toxin type A, B, C, D, E, F or Gpreparation (for example between about 0.1 units and about 50 units of abotulinum toxin type A preparation) is injected directly into one ormore of the parathyroids using one of the techniques set forth inExamples 1 or 2 above. Within 1-7 days the symptoms of the hypocalcemiaare alleviated. Plasma calcium levels return to substantially normallevels. Alleviation of the hypocalcemia persists for at least about 2months to about 6 months.

Additionally, to treat hypercalcemia between about 10⁻³ U/kg and about35 U/kg of a botulinum toxin type A, B, C, D, E, F or G preparation (forexample between about 0.1 units and about 50 units of a botulinum toxintype A preparation) is injected directly into the cervical ganglia asfollows. A percutaneous procedure is carried out using an anteriorapproach with the patient in a supine position using a very thin needle(22 Gauge) with computed tomography needle guidance to reach thecervical ganglia. Within 1-7 days the symptoms of the hypercalcemia arealleviated. Plasma calcium levels return to normal (are decreased).Alleviation of the hypercalcemia persists for at least about 2 to about6 months.

Methods according to the invention disclosed herein has many advantages,including the following:

(1) the invention renders unnecessary many surgical procedures foreffective treatment-of a parathyroid disorder.

(2) systemic drug effects can be avoided by direct local application ofa neurotoxin according to the present invention.

(3) the ameliorative effects of the present invention can persists, onaverage, from about 2 months to about 6 months from a single localadministration of a neurotoxin as set forth herein.

Although the present invention has been described in detail with regardto certain preferred methods, other embodiments, versions, andmodifications within the scope of the present invention are possible.For example, a wide variety of neurotoxins can be effectively used inthe methods of the present invention. Additionally, the presentinvention includes local parathyroid administration methods wherein twoor more neurotoxins, such as two or more botulinum toxins, areadministered concurrently or consecutively. For example, botulinum toxintype A can be administered until a loss of clinical response orneutralizing antibodies develop, followed by administration of botulinumtoxin type E. Alternately, a combination of any two or more of thebotulinum serotypes A-G can be locally administered to control the onsetand duration of the desired therapeutic result. Furthermore,non-neurotoxin compounds can be administered prior to, concurrently withor subsequent to administration of the neurotoxin to proved adjuncteffect such as enhanced or a more rapid onset of denervation before theneurotoxin, such as a botulinum toxin, begins to exert its therapeuticeffect.

My invention also includes within its scope the use of a neurotoxin,such as a botulinum toxin, in the preparation of a medicament for thetreatment of a parathyroid disorder by local administration of theneurotoxin.

Accordingly, the spirit and scope of the following claims should not belimited to the descriptions of the preferred embodiments set forthabove.

I claim:
 1. A method for treating hypocalcemia, the method comprisingthe step of local administration to a parathyroid gland of atherapeutically effective amount of a botulinum toxin, therebyincreasing a parathyroid hormone secretion from a parathyroid cell andtreating hypocalcemia.
 2. The method of claim 1, wherein the botulinumtoxin is selected from the group consisting of botulinum toxin types A,B, C₁, D, E, F and G.
 3. The method of claim 1, wherein the botulinumtoxin is botulinum toxin type A.
 4. A method for treating hypocalcemia,the method comprising the step of local administration to a parathyroidgland of a therapeutically effective amount of a botulinum toxin type A,thereby increasing a parathyroid hormone secretion from a parathyroidcell and treating hypocalcemia.